Birch bark processing and the isolation of natural products from birch bark

ABSTRACT

The invention provides methods for separating outer birch bark from inner birch bark. The invention also provides methods for isolating betulin; lupeol; betulinic acid; 9,10-epoxy-18-hydroxyoctadecanoic acid; 9,10,18trihydroxyoctadecanoic acid; polyphenolic polymers and fatty acids from birch bark.

BACKGROUND OF THE INVENTION

[0001] Birch bark is a low-value waste product in the forest industrytoday. Ekman, R., Holzforschung, (1983) 37, 205. Approximately 230,000tons of birch bark are generated per year. For example, a single papermill can generate 70 tons of birch bark per day. Thus, vast quantitiesof birch bark and its chemical components are available.

[0002] Birch bark is a potential source of a variety of organicchemicals. Several triterpenoids have been identified in birch barkextracts. For example, lupeol, betulin, betulinic aldehyde, betulinicacid, methyl betulinate, lupenone, betulonic aldehyde, betulonic acid,β-amyrin, erythrodiol, oleanolic aldehyde, oleanolic acid, methylleanolate and acetyl oleanolic acid are all present in the outer bark ofBetula verrucosa. Eckerman, C., (1985) Paperi ja Puu, No. 3, 100. Inaddition, several suberinic acids isolated from birch bark, as well asseveral triterpenoids, have been identified in the bark of Betulaverrucosa. Ekman, R., Holzforschung, (1983) 37, 205.

[0003] The chemical constituents of birch bark are useful inpharmaceutical and industrial applications. For example, U.S. Pat. No.5,750,578 discloses that betulin possesses antiviral properties and isuseful to treat herpesvirus. Betulin also possesses antifeedant activityagainst boll weevils, and anti-inflammatory activity (Miles, D. H.,1994, J. Agric. Food. Chem., 42, 1561-1562 and Recio, M., Planta Med.,1995, 61, 9-12. In addition, betulin showed cough suppressant andexpectorant effects. Jinuhua, W., Zhongguo Yaoxue Zazhi, (1994), 29(5),268-71. Betulin is also a useful starting material for preparingalobetulin and derivatives thereof, which posses useful pharmacologicalproperties.

[0004] Betulin can be converted to betulinic acid, which is useful as atherapeutic agent. For example, Pisha, E. et al., (1995) J. M. NatureMedicine, 1, 1046-1051 discloses that betulinic acid has antitumoractivity against human melanoma, e.g., MEL-1, MEL-2 and MEL-4. Inaddition, Fujioka, T. et al., J. Nat, prod., (1994) 57, 243-247discloses that betulinic acid has anti-HIV activity in H9 lymphocyticcells.

[0005] Ambrettolide (cis-hexadec-7-enolide), a naturally occurringcompound, is used to induce musk fragrance in perfumes. Ambrettolide isfound in the vegetable oil of ambrette seeds. The synthesis ofambrettolide is accomplished from 9,10,18-trihydroxyoctadecanoic acidvia a high-yielding multi-step synthesis. Seoane, E., J. Chem. Soc.Perkin Trans. (1982), 1837-1839. Therefore,9,10,18-trihydroxyoctadecanoic acid, which is present in birch bark, isa useful precursor for the synthesis of ambrettolide.

[0006] 9,10-Epoxy-18-hydroxyoctadecanoic acid is also present in birchbark. 9,10-Epoxy-18-hydroxyoctadecanoic acid is anenvironmentally-friendly spoilage deterrent and a rot-resistant additivefor wood composites. Sweitzer, P., et al., Induction of Resistance inBarley Against Erysiphe graminis by Free Cutin Monomers, Physiol. Mol.Plant Pathol, (1996) 49(2) 103-120.

[0007] Suberin is another major component of birch bark. Suberin is aninsoluble polymeric material that is attached to the cell walls ofperidenms. Kola, P. E. et al., Ann. Rev. Plant. Physiol., (1981),32:539-67. Suberin is generally an ester of fatty acids and polyphenolicpolymers. Suberin of birch bark is typically a biopolyester of primaryhydroxy, epoxy and dicarboxylic acids. Ekman, Holzforschung, (1983) 37,205-211.

[0008] Suberin posseses several industrial applications. See, e.g.,Taylor and Francis, Forests Products Biotechnology, A. Bruce and J. W.Palfreyman (editors), 167, 179-181 (1998); Peter E. Laks and Peggy A.McKaig, Flavonoid Biocides: Wood Preservatives Based on CondensedTannins, Hlorzforschung, 42, 299-306 (1988); Etherington & Roberts,Dictionary —birch(bark),http://sul-server-2.stanford.edu/don/dt/dt0328.html, 1, Jun. 23, 1999;P. E. Kolattukudy, Structure, Biosynthesis, and Biodegradation of Cutinand Suberin, Ann. Rev. Plant Physiol., 32, 539-67 (1981); and N.Cordeiro, M. N. Belgasem, A. J. D. Silvestre, C. Pascol Neto, A.Gandini, Cork Suberin as a new source of chemicals, Int. Journal ofBiological Materials, 22, 71080 (1998). Suberin is useful as adispersant in many industrial applications (e.g., carbon black slurries,clay products, dyes, cement, oil drilling muds, and asphaltemulsifiers). Suberin is also useful in binders for animal pellets,conditioners for boiling water, anti-oxidants and additives tolead-storage battery plate expanders. McGraw-Hill Concise Encyclopediaof Science & Technology, Fourth Edition, 1998.

[0009] Polyphenolic polymers are also present in birch bark as aconstituent of suberin. Polyphenolic polymers may be classified assoluble polyphenolic polymers or non-soluble polyphenolic polymers.Soluble polyphenolic polymers are the portion of polymers which aresoluble in water under both acidic and basic conditions. The non-solublepolyphenolic polymers are non-soluble in water at a pH below about 4.0,but soluble in acetone, alcohols and other polar solvents. Thenon-soluble polyphenolic polymers may have a different formulation fromthe soluble polyphenolic polymers. However, the non-soluble polyphenolicpolymers may be used in the same industrial applications as the solublepolyphenolic polymers.

[0010] Polyphenolic polymers are non-toxic and biodegradable and may beformulated for numerous purposes (e.g., as anti-oxidant reagents,anti-flngal materials, coating materials, co-polymers, woodpreservatives, tire cord adhesives, foundry cord binders, rigid andfloral foams, ion exchange resins, industrial water purificationflocculants, textile dyes, food additives and pharmaceuticals). Pizzi,Wood Bark Extracts as Adhesives and Preservatives, 167-181, Taylor &Frances, Forest Products Biotechnology, Bruce and Palfreyman (editors),1998.

[0011] Current methods for isolating the chemical constituents of birchbark are deficient in several ways. For example, betulin has beenextracted from the bark of white-barked birches in amounts up to 30%,based on the dry weight of the bark. Elkman, R., (1983) Holzforsch, 37,205; Ohara, S., et al., (1986) Mokuza Gakkaishi, 32, 266. In addition,Betulin has been isolated from outer birch bark waste of Betulaverrucosa by liquid extraction employing boiling organic solvents andsubsequent recrystallization. Eckerman, C., (1985) Paperi ja Puu, No. 3,100. While current processes afford acceptable yields of betulin (e.g.,11-30%), these processes suffer from several major drawbacks. Forexample, the use of a boiling organic solvent, at standard pressure, inthe extraction of betulin may destroy other useful compounds present inthe bark. A need therefore exists for a method that can be used toextract betulin without damaging other compounds remaining in the birchbark.

[0012] Another drawback with the current extraction processes is thatthe organic solvents employed are hazardous, difficult to handle ordifficult to dispose of. The typical organic solvents, which includemethylene chloride and chloroform, are hazardous to humans (i.e., theyare toxic or carcinogenic) and are hazardous to the environment.Considering the industrial scale on which the extraction processes wouldneed to be performed in order to provide industrial quantities (e.g.,tons) of betulin, large quantities of organic solvents would berequired. The high cost of disposing the organic solvents is anadditional disadvantage of the current extraction procedures.

[0013] Several methods have been devised for isolating polyphenolicpolymers from birch trees. Some isolation methods are based on acidtreatments in which the carbohydrate components (cellulose andhemicelluloses) are hydrolyzed to water-soluble materials. However, withsuch procedures, serious doubts exist as to whether the isolatedpolyphenolic polymer is representative of the “native” polyphenolicpolymer. In addition, extraction conditions can cause undesirablerearrangements and other transformations of the polyphenolic structurethat lead to a loss of useful properties. It is therefore desirable tohave polyphenolic polymers in a form in which it is readily accessible,without involving costly, lengthy or dangerous procedures.

[0014] Suberin from betula verucosa contains at least 35 fatty acidswhich makes it hardly usable in industry. U.S. Pat. No. 4,732,708 issuedto Ekman, R. et al. discloses a process for manufacturing suberinicacid. The process, however, does not attempt to separate the individualfatty acids. In addition, due to the crucial differences in thefundamental chemistry between the types of birch trees (i.e., the typeand distribution of fatty acids), the procedures employed in U.S. Pat.No. 4,732,708 issued to Ekman, R. et al. may not be useful for theisolation of fatty acids from species of birch bark other than thoseemployed in U.S. Pat. No. 4,732,708. As such, a method for isolating theindividual fatty acids from the bark of other species of birch isneeded.

[0015] The current methods employed to isolate not only betulin, butother components in birch bark (e.g., lupeol; betulinic acid;9,10-epoxy-18-hydroxyoctadecanoic acid; 9,10,18-trihydroxyoctadecanoicacid; and polyphenolic polymers) are costly, inefficient or unsafe. Aneed therefore exists for safer, more cost-efficient methods to obtaincommercial quantities (e.g., tons) of betulin; as well as commercialquantities (e.g., kg) of lupeol; betulinic acid;9,10-epoxy-18-hydroxyoctadecanoic acid; 9,10,18-trihydroxyoctadecanoicacid; and polyphenolic polymers from birch bark. In addition, a needalso exists for an industrial scale process for producing theseproducts.

SUMMARY OF THE INVENTION

[0016] The present invention provides methods for isolating the chemicalconstituents of birch bark. Specifically, the present invention providesa method that can be used to extract betulin from birch bark withoutdamaging other compounds remaining in the birch bark. In addition, theextraction processes employ solvents that are safe (non-toxic andnon-carcinogenic), easy to handle, environmentally-friendly,inexpensive, and easy to dispose of. The present invention also providesa method for isolating polyphenolic polymers from birch trees whereinthe polyphenolic polymers are in a form that is readily accessible andthe methods do not involve costly, lengthy or dangerous procedures. Thepresent invention also provides a method for isolating the individualfatty acids from the bark of various species of birch. The presentinvention also provides methods to provide commercial quantities (e.g.,tons) of betulin; as well as commercial quantities (e.g., kg) of lupeol;betulinic acid; 9,10-epoxy-18-hydroxyoctadecanoic acid;9,10,18-trihydroxyoctadecanoic acid; and polyphenolic polymers frombirch bark.

[0017] The present invention provides a process for separating outerbirch bark from inner birch bark comprising subjecting birch bark tofragmentation to provide a combination of outer birch bark shreds andinner birch bark chunks and separating the outer birch bark shreds fromthe inner birch bark chunks.

[0018] The present invention also provides a process that provides oneor more (e.g., 1, 2, 3, or 4) natural products from outer birch bark.Accordingly, there is provided a process for obtaining one or morenatural products from outer birch bark comprising subjecting the outerbirch bark to supercritical fluid extraction to provide the naturalproduct.

[0019] The present invention also provides a process for obtaininglupeol, betulinic acid and betulin from outer birch bark comprisingextracting outer birch bark with carbon dioxide at a pressure betweenabout 3,000 psi and 10,000 psi and at a temperature between about 50° C.and 100° C. to provide lupeol, betulin and betulinic acid.

[0020] The present invention also provides a process for obtaininglupeol, betulinic acid and betulin from outer birch bark usingfractional supercritical fluid extraction comprising extracting withcarbon dioxide at a pressure below about 5,000 psi and at a temperaturebelow about 50° C. to provide a product comprising lupeol and extractingwith carbon dioxide at a pressure of about 5,000 psi to about 10,000 psiand at a temperature of about 50° C. to about 120° C. to provide aproduct comprising a mixture of betulin and betulinic acid.

[0021] The present invention also provides a process for obtaininglupeol from outer birch bark comprising subjecting the outer birch barkto supercritical fluid extraction with carbon dioxide at a temperatureof about 40° C. to about 50° C. and a pressure of about 3,000 psi toabout 5,000 psi for a period of time of about 1 hour to about 3 hours toprovide the lupeol.

[0022] The present invention also provides a process for obtainingbetulin and betulinic acid from outer birch bark comprising subjectingthe outer birch bark to supercritical fluid extraction with carbondioxide at a temperature of about 80° C. to about 100° C. and a pressureof about 8,000 psi to about 10,000 psi for a period of time of about 3hours to about 5 hours to provide a mixture of betulin and betulinicacid.

[0023] The present invention also provides a process for isolating9,10-epoxy-18-hydroxyoctadecanoic acid from outer birch bark comprising:(1) subjecting the outer birch bark to alkali hydrolysis in an aqueousalcohol solution to provide a second outer birch bark and a secondsolution; (2) separating the second solution from the second outer birchbark; (3) condensing the second solution at a temperature below about50° C. to form a third solution; (4) adding water to the third solutionto form a precipitate and a fourth solution; (5) separating theprecipitate from the fourth solution; (6) acidifying the fourth solutionto a pH of about 5.5 to about 6.5 to give a fifth solution and a secondprecipitate; (7) separating the second precipitate from the fifthsolution (8) condensing the fifth solution to provide a sixth solution;(9) subjecting the sixth solution to epoxidizing conditions to providean epoxide and hydrolyzing the epoxide to provide a seventh solution;and (10) crystallizing the seventh solution to give9,10,18-trihydroxyoctadecanoic acid.

[0024] The present invention also provides a process for isolatingnon-soluble polyphenolic polymers and fatty acids from outer birch barkcomprising: (1) subjecting the outer birch bark to alkali hydrolysis inan aqueous alcohol solution to provide a second birch bark and a secondsolution; (2) separating the second solution from the second outer birchbark; (3) adding water to the second outer birch bark to provide a thirdsolution and a third outer birch bark; (4) separating the third solutionfrom the third outer birch bark; (5) acidifying the third solution to apH of about 3.0 to about 4.0 to give a fourth solution and a mixture ofnon-soluble polyphenolic polymer and fatty acids; and (6) separating themixture of fatty acids and non-soluble polyphenolic polymers from thefourth solution.

[0025] The present invention also provides a process for isolating fattyacids and soluble polyphenolic polymers from outer birch barkcomprising: (1) subjecting the outer birch bark to alkali hydrolysis inan aqueous alcohol solution to provide a second outer birch bark and asecond solution; (2) separating the second solution from the secondouter birch bark; (3) adding water to the second outer birch bark toprovide a third outer birch bark and a third solution; (4) separatingthe third solution from the third outer birch bark; (5) acidifying thethird solution to a pH of about 3.0-4.0 to give a fourth solution and asolid; (6) separating the solid from the fourth solution; (7) adding analcohol to the fourth solution to provide a fifth solution and aprecipitate; (8) separating the precipitate from the fifth solution; and(9) condensing the fifth solution to provide a mixture of fatty acidsand soluble polyphenolic polymers.

BRIEF DESCRIPTION OF THE FIGURES

[0026]FIG. 1(a) illustrates outer and inner birch bark (cross-sectionalview).

[0027]FIG. 1(b) illustrates outer and inner birch bark.

[0028]FIG. 2 illustrates an apparatus for supercritical fluidextraction.

[0029]FIG. 3 is a schematic illustration of the isolation of lupeol,betulin and betulinic acid from outer birch bark.

[0030]FIG. 4 is a schematic illustration of the isolation of9,10-epoxy-18-hydroxyoctadecanoic acid from outer birch bark.

[0031]FIG. 5 is a schematic illustration of the isolation of9,10,18-trihydroxyoctadecanoic acid from outer birch bark.

[0032]FIG. 6 is a schematic illustration of the isolation of a mixtureof fatty acids and non-soluble polyphenolic polymers (NPPP) from outerbirch bark.

[0033]FIG. 7 is a schematic illustration of the isolation of a mixtureof fatty acids and soluble polyphenolic polymers (SPPP) from outer birchbark.

[0034]FIG. 8 is a schematic drawing of the isolation of lupeol; betulin;betulinic acid; 9,10-epoxy-18-hydroxyoctadecanoic acid;9,10,18-trihydroxyoctadecanoic acid; a mixture of fatty acids andnon-soluble polyphenolic polymers (NPPP); and a mixture of fatty acidsand soluble polyphenolic polymers (SPPP) from outer birch bark.

[0035]FIG. 9 illustrates the separation of outer and inner bark with anair classifier.

DETAILED DESCRIPTION OF THE INVENTION

[0036] Specific values listed below for ranges are for illustrationonly; they do not exclude other defined values or other values withindefined ranges.

[0037] Separation Of Inner Birch Bark from Outer Birch Bark

[0038] As used herein, “birch” is any of the several deciduous trees ofthe genus Betula. The birches comprise the family Betulaceae in theorder Fagales. Birch trees include, for example, white birch, B. alba;sweet, black or cherry birch, B. lenta; monarch birch, B.Maximowicziana; dwarf or arctic birch, B. Nana; Japanese white birch, B.Platyphyla Japonica; smooth-bark birch, B. Pubescens; yellow birch, B.alleghaniensis; paper, white or canoe birch, B. papyrifera; grey birch,B. populifolia; river birch, B. nigra; and the European birches, B.pubescens; B. Alba and B. pendula. Specifically, birch can be B. alba,B. Jenta, B. Maximowicziana, B. Nana, B. Platyphyla Japonica, B.Pubescens, B. alleghaniensis, B. papyrifera, B. popuhifolia, B. nigra,B. pubescens, B. Alba or B. pendula. A specific birch for use in theprocesses of the present invention is B. papyrifera.

[0039] As used herein, “fragmentation” includes chopping, crunching,crushing, gnashing or pounding. Such fragmentation of birch bark willeffectively provide inner birch bark (e.g., in the form of chunks) andouter birch bark (e.g., in the form of shreds) which can be physicallyseparated. The fragmentation can conveniently be carried out by feedingbirch bark into a machine with knives on a rotating disk (e.g., achipper or shredder). One chipper suitable for fragmenting the bark isthe YardMan Model 246-648D401 chipper.

[0040] As illustrated in FIG. 1(a) and FIG. 1(b), birch bark consists ofinner birch bark and outer birch bark. Inner birch bark is more denseand granular than outer birch bark, while outer birch bark is moreflexible and fibrous than inner birch bark. In addition, outer birchbark is light in color, thin (1-5 mm), tough, and of low water-contentrelative to inner birch bark. The inner bark is darker in color, thicker(3-10 mm) and non-fibrous relative to the outer bark. The inner bark isthe portion of the tree wherein significant water transport occurs(i.e., an area of high water content). Due to the differences in thephysical properties of inner birch bark and outer birch bark, Applicanthas found that fragmentation produces outer birch bark shreds and innerbirch bark chunks.

[0041] Outer birch bark shreds can be separated from the inner birchbark chunks using any suitable means. The separation can conveniently beaccomplished by screening the mixture through a mesh having openingsintermediate in size between the smaller inner bark chunks and thelarger outer bark shreds. The smaller inner bark chunks fall through thescreen and are separated from the outer bark.

[0042] The “mesh” can be a unit comprising one or more open spaces in acord, thread, or wire network in which the cords, threads or wiressurround the spaces. Any mesh suitable to separate inner birch bark fromouter birch bark can be employed. Typically, the mesh is a wire meshcontaining openings of about ½ of an inch by ½ of an inch, or smaller.

[0043] For example, mesh can conveniently contain openings of about ¼ ofan inch by about ¼ of an inch. Specifically, the size of the mesh can beabout 20 mm by about 20 mm, or about 10 mm by about 10 mm, or about 6 mmby about 6 mm. More specifically, the size of the mesh can be about 3 mmby about 3 mm.

[0044] Alternatively, the inner birch bark chunks and outer birch barkshreds may be separated with the use of an air classifier as shown inFIG. 9. As used herein, an “air classifier” is a device which operateson the principle of the differing properties of the two components(e.g., inner and outer birch bark) in an air stream to effect a physicalseparation. Typically, the less dense outer bark travels a greaterdistance in the air stream than the more dense inner bark. The innerbark, along with other materials, falls rapidly from the stream of air.As a result, the inner birch bark and the outer birch bark can beseparated.

[0045] After separating outer birch bark from inner birch bark, outerbirch bark of about 10 wt.% to about 45 wt.% based on initial birch barkcontent is typically obtained and inner birch bark of about 55 wt.% toabout 85 wt.% is typically obtained.

[0046] For use in the processes of the present invention, birch barkshreds less than about 10 mm in diameter can conveniently be used. Morespecifically, outer birch bark shreds less than about 6 mm in diameter,less than about 4 mm in diameter, or less than about 2 mm in diameter,can be used.

[0047] Supercritical Fluid Extraction

[0048] As illustrated in FIG. 2, a natural product (e.g., betulin,betulinic acid or lupeol) can be obtained from outer birch bark bysupercritical fluid extraction. The outer birch bark is introduced intoa feed tank (1) through the opened lid on the top. The birch bark isheated at an elevated pressure in a solvent comprising carbon dioxide.The solution is transferred to a product reservoir (2). Extractedproduct is removed and the solvent comprising carbon dioxide is passedthough a condenser (3) and subsequently recycled into the feed tank (1)through a recycler (4).

[0049] As used herein, “natural product” is any of the compoundsnaturally occurring in the bark of birch. Natural products specificallyinclude triterpenoids.

[0050] According to the biogenetic isoprene rule, a “triterpenoid” is ahydrocarbon, or its oxygenated analog, that is derived from squalene bya sequence of straightforward cyclizations, functionalizations, andsometimes rearrangement.

[0051] A specific triterpenoid present in birch bark is betulin(lup-20(29)ene-3,28-diol), lupeol (lup-20(29)-en-3β-ol), or betulinicacid (lup-20(29)-en-3β-ol-28-oic acid).

[0052] The processes of the present invention provide natural productsof birch bark. Each natural product may have one or more chiral centersand may exist in and be isolated in optically active and racemic forms.It is to be understood that the present invention provides processes forisolating natural products in any racemic, optically-active,polymorphic, or stereoisomeric form, present in the native bark orisolated after exposure to the processes of the invention. When aprocess of the invention provides a mixture of enantiomers or isomers,it is appreciated that those skilled in the art can separate opticallyactive forms (for example, by resolution of the racemic form byrecrystallization techniques or by chromatographic separation using achiral stationary phase) if a single enantiomer is desired.

[0053] Supercritical fluid extraction is an extraction wherein a fluidat a temperature and pressure above its critical point is employed; or afluid above its critical temperature, regardless of pressure, isemployed. Below the critical point, the fluid can coexist in both gasand liquid phases, but above the critical point there is only one phase.Equipment and techniques for carrying out supercritical fluid extractionare known to those skilled in the art. See, McHugh, M. And Krnkonis, V.,Supercritical Fluid Extraction, 2nd ed, Butterworth-Heinemann, Boston,1994; Johnston, K. P., Penninger, J. M. L., Supercritical Fluid Scienceand Technology, ACS Symposium Series 406, American Chemical Society,Washington, D.C.; and Taylor, L. T., Supercritical Fluid Extraction,John Wiley & Sons, N.Y., 1996.

[0054] In a supercritical fluid extraction, thermodynamic and transportproperties of supercritical fluid are a function of density, whichdepends strongly on the fluid's pressure and temperature. The densitymay be adjusted from a gas-like value of 0.1 g/ml to a liquid-like valueas high as 1.2 g/ml. Furthermore, as conditions approach the criticalpoint, the effect of temperature and pressure on density becomes muchmore significant. For example, increasing the density of supercriticalcarbon dioxide from 0.2 to 0.5 g/ml requires raising the pressure from85 atm to 140 atm (8.6 megapascals to 14.2 megapascals) at 158° F. (70°C.), but at 95° F. (35° C.) the required change is only from 65 atm to80 atm (6.61 Mpa to 8.1 Mpa).

[0055] As used herein, “fractional supercritical fluid extraction”(hereinafter “FSCFE”) is a multi-step procedure wherein thesupercritical fluid extraction is carried out at one temperature andpressure for a given period of time and is then carried out at one ormore other temperatures or pressures.

[0056] The efficiency of supercritical fluid extraction on a materialsuch as outer birch bark depends in part upon the size of the outerbirch bark pieces. Thus, the smaller the outer birch bark pieces, themore efficient the supercritical fluid extraction typically will be. Assuch, after fragmentation and prior to extraction, outer birch barkshreds may be further reduced in size with a Hammermill or suitablemeans. For example, a 15 horsepower 3B Junior Hammermill made by Jay BeeManufacturing, Inc can be used as illustrated in the Examples hereinbelow. The hammernill reduces large pieces of birch bark by beating thebark with pivoted hammers until the material is small enough to fallthrough a mesh.

[0057] For use in the processes of the present invention, the size ofouter birch bark shreds obtained after the Hammermill reduction istypically less than about 5 mm in diameter. Specifically, the shreds canbe less than about 3 mm in diameter. More specifically, the shreds canbe less than about 1 mm in diameter.

[0058] Prior to fragmentation or extraction, outer birch bark may bedried of any water present. Such drying may increase the efficiency ofthe fragmentation. Birch bark may be air-dried or dried at an elevatedtemperature with or without reduced pressure (i.e., in vacuo).Specifically, birch bark may be dried in vacuo at an elevatedtemperature. Machines capable of drying bark are known in the art andinclude an oven, or similar device, such as a rotating air drum drier.

[0059] For use in the processes of the present invention, supercriticalfluid extraction can conveniently be carried out at a pressure of about1,000 psi to about 12,000 psi. It is appreciated that those skilled inthe art understand that higher pressures may enable faster extraction.In this case, it may be necessary to subsequently separate and purifythe product (e.g., lupeol, betulin, betulinic acids, other minortriterpene or acidic admixtures).

[0060] For use in the processes of the present invention, supercriticalfluid extraction can conveniently be carried out at a pressure of about750 psi to about 12,000 psi. Specifically, the pressure may be about1,000 psi to about 10,000 psi. More specifically, the pressure may beabout 4,000 psi to about 9,000 psi.

[0061] For use in the processes of the present invention, thetemperature at which the birch bark may be dried is greater than about30° C. Specifically, the temperature is greater than about 45° C. Morespecifically, the temperature is greater than about 60° C.

[0062] For use in the processes of the present invention, thetemperature of supercritical fluid extraction can conveniently be about0° C. to about 150° C. Specifically, the temperature can be about 25° C.to about 110° C. More specifically, the temperature can be about 45° C.to about 100° C.

[0063] In one specific embodiment, supercritical fluid extraction isperformed at a temperature of about 40° C. to about 90° C. and apressure of about 3,000 psi to about 10,000 psi.

[0064] Supercritical fluid extraction employs a solvent which possessesphysical properties suitable as a supercritical fluid. Suitable solventsinclude carbon dioxide, Xe, Freon-23, ethane, N₂O, SF₆, propane,ammonia, n-C₄H₁₀, (C₂H₅)₂O and the like.

[0065] The physical properties of carbon dioxide make it particularlyattractive as a solvent for supercritical fluid extraction. Carbondioxide is a major component of the atmosphere and is thereforerelatively safe and abundant. In addition, carbon dioxide is relativelyinexpensive. Compared to most other suitable solvents, carbon dioxide isenvironmentally friendly as it will not harm the atmosphere at thequantities used in the methods of the invention. Moreover, carbondioxide is non-flammable and non-explosive. Further, carbon dioxideleaves no substantial residue or remnant upon evaporation.

[0066] Carbon dioxide also possesses physical properties which enable itto change polarity over the temperature range and pressure rangenormally employed in supercritical fluid extraction. As a result, carbondioxide may act as a nonpolar solvent at one temperature and pressurebut may act as a polar solvent at another temperature and pressure. Byvarying the temperature and pressure, the solvent properties may bemodified. This allows for the isolation of more than one compound usinga single solvent system.

[0067] The solvent employed in supercritical fluid extraction may be asingle compound or may be a mixture of compounds. In addition, thesolvent may include an additive.

[0068] As used herein, an “additive” is a compound added to the solventin an amount of about 1 wt% to about 20 wt.% based on the solvent.Specifically, the additive may be present in an amount of about 1 wt.%to about 15 wt.% or about 1 wt.% to about 10 wt.%. Upon addition, theadditive will modify the physical properties of the solvent. Forexample, an additive may be useful to modify the polarity, criticaltemperature, critical pressure, etc., of the solvent system.

[0069] Suitable additives include lower alcohols (e.g., methanol,ethanol, 1-propanol, 2-propanol, 1-hexanol, or 2-methoxy ethanol);ethers (e.g., tetrahydrofuran or 1,4-dioxane); substituted hydrocarbons(e.g., acetonitrile, dichloromethane, ammonia or chloroform) propylenecarbonate, N,N-dimethylaceamide; dimethyl sulfoxide; carboxylic acids(e.g., formic acid); water; carbon disulfide; lower ketones (e.g.,acetone), hydrocarbons (e.g., propane, toluene, hexanes and pentanes).

[0070] Applicant has found that utilizing a solvent comprising carbondioxide in supercritical fluid extraction, lupeol is soluble at atemperature below about 50° C. and a pressure below about 5,000 psi. Inaddition, Applicant has found that following removal of lupeol, betulinand betulinic acid can be extracted using a solvent comprising carbondioxide at a temperature of about 50° C. to about 120° C. and a pressureof about 5,000 psi to about 10,000 psi.

[0071] Accordingly, the processes of the invention can convenientlyinclude a fractional supercritical fluid extraction of outer birch barkemploying a solvent comprising carbon dioxide at a pressure below about5,000 psi at a temperature below about 50° C. to provide lupeol followedby an extraction employing a solvent comprising carbon dioxide at apressure of about 5,000 psi to about 10,000 psi and at a temperature ofabout 50° C. to about 120° C. to provide betulin and betulinic acid.

[0072] The processes of the invention can conveniently include afractional supercritical fluid extraction comprising extracting outerbirch bark with a solvent comprising carbon dioxide for a period of timegreater than about 30 minutes at a pressure below about 5,000 psi and ata temperature below about 50° C. to provide lupeol followed byextracting with a solvent comprising carbon dioxide for a period of timegreater than about 30 minutes at a pressure of about 5,000 psi to about10,000 psi and at a temperature of about 50° C. to about 120° C. toprovide a mixture of betulin and betulinic acid.

[0073] The processes of the invention can conveniently include afractional supercritical fluid extraction comprising extracting with asolvent comprising carbon dioxide for a period of time of about 1 hourto about 3 hours at a pressure of about 3,000 psi to about 5,000 psi andat a temperature of about 40° C. to about 50° C. to provide lupeol andbetulin followed by extracting with a solvent comprising carbon dioxidefor a period of time of about 3 hours to about 5 hours at a pressure ofabout 8,000 psi to about 10,000 psi and at a temperature of about 80° C.to about 100° C. to provide a mixture of betulin and betulinic acid.

[0074] The processes of the invention can also include non-fractionalsupercritical fluid extraction comprising extracting with a solventcomprising carbon dioxide. The extraction can conveniently be carriedout for a period of time of about 3 hours to about 5 hours at a pressureof about 5,000 psi to about 10,000 psi and at a temperature of about 80°C. to about 100° to provide a mixture of lupeol, betulin and betulinicacid.

[0075] Betulin

[0076] The betulin from the mixture of lupeol, betulin and/or betulinicacid obtained from supercritical fluid extraction may be purified usingtechniques that are known in the art for purification of naturalproducts, e.g., by recrystallization or by chromatography. Suitablesolvents for crystallization of betulin include, for example, an alcohol(e.g., isopropanol). After purification, betulin is typically at least90% pure. Specifically, betulin is at least 95% pure, or at least 97%pure.

[0077] Typically, the processes of the present invention provide betulinin a yield of about 10 wt.% to about 25 wt.% based on outer birch bark.Specifically, the yield is about 15 wt.% to about 20 wt.% based on outerbirch bark, or about 17 wt.% to about 20 wt.% based on outer birch bark.

[0078] Using the procedures of the present invention, betulin can berecovered in about 15 wt.% to about 25 wt.%, based on birch bark. Thisrepresents as much as a 25% improvement over other methods for betulinisolation. O'Connell, M. M.; Bentley, M.D.; Campbell, C. S.; Cole, B. J.W.; Phytochemistry (1988) 27, 2175-2176; Lugemwa, F. N.; Huang, F. Y.;Bentley, M.D.; Mendel, M. J.; Alford, A. R.; J. Agric. Food Chem.,(1990), 36, 493-496.

[0079] Betulinic Acid

[0080] Betulinic acid may be separated and purified through the specificformation of non-soluble aluminum salts of betulinic acid with aluminumalcoholates. As used herein, “alcoholate” is an organic alcohol whereinthe hydroxy hydrogen has been replaced with a metal, e.g., (CH₃CH₂O)₃Al.Aluminum alcoholates are suitable reagents for triterpene purificationbecause it is believed that aluminum alcoholates bind strongly andirreversibly to acids and tannins, therefore providing completediscoloration of the total extract.

[0081] A suitable aluminum alcoholate is aluminum isopropoxide, however,other alcoholates, basic materials or ion exchange resins may beemployed to purify the betulinic acid.

[0082] Further purification of betulinic acid from aluminum salts may beprovided using techniques that are known in the art for the purificationor isolation of natural products, e.g., by washing with solvent,crystallization, using ion exchange resins, through the formation ofesters or by chromatography. After purification, betulinic acid istypically at least 90% pure. Specifically, betulinic acid is at least95% pure, or at least 99% pure.

[0083] The processes of the present invention yield betulinic acid,after purification, that is about 0.5 wt.% to about 2 wt.% based onouter birch bark. Specifically, betulinic acid is about 1 wt.% to about1.5 wt.% based on outer birch bark, or about 0.5 wt.% to about 1 wt.%based on outer birch bark.

[0084] Although betulinic acid is reported as being present in birchbark, the isolation, separation and purification of betulinic acid frombirch bark has not been previously reported. The processes of thepresent invention yield betulinic acid of about 0.5 wt.% to 2 wt.% basedon the outer bark of betula paparifera. In addition, the processes ofthe present invention may be used to isolate betulinic acid present inother kinds of birch bark. Moreover, the processes of the presentinvention (e.g., SCFE) may be used to separate and isolate betulinicacid from other kinds of plant extracts.

[0085] Lupeol

[0086] The processes of the present invention yield lupeol, afterpurification, that is about 0.5 wt% to about 2 wt.% based on outer birchbark. Specifically, the lupeol is about 1wt.% to about 1.5 wt.% based onouter birch bark, or about 0.5 wt.% to about 1.0 wt.% based on outerbirch bark.

[0087] The lupeol obtained from the supercritical fluid extraction mayalso be purified using techniques that are known in the art forpurification of natural products, e.g., by recrystallization or bychromatography on silica gel or other suitable supports. Suitablesolvents for chromatographic purification of lupeol include, forexample, a non-polar solvent (e.g., hexanes:ether, 4:1). Afterpurification, lupeol is typically at least 90% pure. Specifically,lupeol is at least 95% pure, or at least 98% pure.

[0088] Using the procedures of the present invention, lupeol can berecovered in about 0.5 wt.% to about 2 wt.%, based on birch bark. Thisrepresents as much as a 70% improvement over other methods for lupeolisolation. O'Connell, M. M.; Bentley, M.D.; Campbell, C. S.; Cole, B. J.W.; Phytochemistry (1988) 27, 2175-2176; Lugemwa, F. N.; Huang, F. Y.;Bentley, M.D.; Mendel, M. J.; Alford, A. R.; J. Agric Food Chem.,(1990), 36, 493-496.

[0089] Applicant has discovered that betulin, lupeol and betulinic acidmay be isolated from outer birch bark in good yield and purity, usingsupercritical fluid extraction. Conditions previously known forisolating one or more of these compounds damage much of the suberinpresent in the bark. SCFE, however, is a milder technique which does notdestroy the oxirane rings of suberin.

[0090] Isolation of 9,10-Epoxy-18-Hydroxydecanoic Acid (FIG. 4)

[0091] As illustrated in FIG. 4, step 1, outer birch bark is subject toalkali hydrolysis. As used herein, “alkali hydrolysis” includes anycondition suitable for saponifying ester bonds. The reaction (i.e.,alkali hydrolysis) can conveniently be carried out in an aqueous alcoholsolution under basic catalysis.

[0092] As used herein, “alcohol” is a compound containing at least oneC(OH) group. Particular alcohols for use in the present invention willhave between about 1 and about 10 carbon atoms; may be cyclic oraliphatic; may be saturated or unsaturated; and may be branched orstraight-chained. Specific alcohols suitable for use in the presentinvention include methanol, ethanol, iso-propanol, tert-butanol,1-hepten-3-ol and 1-octen-3-ol.

[0093] As used herein, “aqueous alcohol solution” is a solutioncomprising water and an alcohol. Typically, the alcohol is present in atleast 20, 50, or 75 wt.% of the solution. Specifically, the alcohol ispresent in about 90 wt.% of the solution or in about 95 wt.% of thesolution. As illustrated in the Examples herein below, a specificaqueous alcohol solution suitable for use in the processes of thepresent invention is about 5% water in isopropanol.

[0094] Suitable bases include metal hydroxides and metal alkoxides.Suitable metal hydroxides include sodium hydroxide, potassium hydroxide,lithium hydroxide, calcium hydroxide and barium hydroxide. Suitablemetal alkoxides include lithium methoxide, lithium ethoxide, lithiumisopropoxide, lithium tert-butoxide, sodium methoxide, sodium ethoxide,sodium isopropoxide, sodium tert-butoxide, potassium methoxide,potassium ethoxide, potassium isopropoxide, potassium tert-butoxide,magnesium methoxide, magnesium ethoxide, barium methoxide, bariumethoxide, calcium methoxide and calcium ethoxide.

[0095] A specific base suitable for the processes of the presentinvention is sodium hydroxide.

[0096] Prior to alkali hydrolysis, outer birch bark can optionally besubject to extraction as illustrated in FIG. 2 and FIG. 3 to provideextracted outer birch bark. As used herein, “extraction” is the act ofobtaining one or more compounds by chemical or mechanical action, as bypressure, distillation, or evaporation as described herein above.Extraction includes the use of a solvent, for example, water or anorganic solvent, at standard temperature and pressure. In addition,extracting also includes supercritical fluid extraction.

[0097] Extracting outer birch bark is useful to remove compounds thatmay interfere with the subsequent hydrolysis, or that may contaminatethe hydrolysis product. For example, lupeol and betulin are optionallyremoved from the outer birch bark prior to the outer birch bark beingsubject to alkali hydrolysis to facilitate the isolation of9,10-epoxy-18-hydroxyoctadecanoic acid from outer birch bark.

[0098] As illustrated in FIG. 4, step 2, the second solution (i.e.,aqueous alcohol solution) is separated from the second outer birch bark.The solution can be separated using any suitable technique for removinga solid from a liquid. For example, the separation can be accomplishedby filtering, hot filtering, or centrifuging. Specifically, the secondsolution can be separated from the second outer bark by filtering, andmore specifically, by hot filtering. “Hot filtering” includes filteringa solid from a liquid wherein both the solid and liquid, prior tofiltering, are at a temperature above about 40° C. Specifically, thetemperature is above about 55° C. More specifically, the temperature isabove about 70° C.

[0099] As illustrated in FIG. 4, step 3, the second solution iscondensed at a temperature below about 50° C. to provide a thirdsolution. The solution can be condensed using any suitable techniquethat is known in the art. For example, “condensing” can includeevaporating or evaporating in vacuo. Specifically, condensing can occurby evaporating in vacuo at a temperature less than about 50° C. orevaporating in vacuo at a temperature less than about 35° C. Morespecifically, condensing can occur by evaporating in vacuo at atemperature less than about 30° C. Condensing can conveniently becarried out for a period of time sufficient to reduce the volume of thesolution at least about 20%, 50%, 75% or 90%.

[0100] Applicant has found that the temperature of condensing (e.g.,evaporation) influences the ability to isolate9,10-epoxy-18-hydroxyoctadecanoic acid from outer birch bark. Applicanthas found that the temperature during condensing preferably should bekept below about 50° C. If the temperature during condensing is keptbelow about 50° C., 9,10-epoxy-18-hydroxyoctadecanoic acid will notreadily decompose to 9,10,18-trihydroxyoctadecanoic acid, and a higheryield of 9,10-epoxy-18-hydroxyoctadecanoic acid can be obtained.

[0101] As illustrated in FIG. 4, step 4 and step 5, water, or anothersuitable solvent, is added to the third solution to form a precipitateand a fourth solution, and the precipitate is separated from the fourthsolution. The precipitate can be separated using any suitable techniqueknown in the art. For example, it may be separated by filtering, hotfiltering, or centrifuging. However, the precipitate typically has aclay-like form. As such, filtration of the precipitate can be extremelydifficult. However, the precipitate can conveniently be separated fromthe fourth solution by centrifuging.

[0102] As illustrated in FIG. 4, step 6, the fourth solution isacidified to a pH of about 5.5 to about 6.5 to give a fifth solution and9,10-epoxy-18-hydroxydecanoic acid (i.e., oxirane) as a precipitate. ThepH of the fourth solution may be lowered by the addition of a suitableacid. Suitable acids include, for example, hydrochloric acid, phosphoricacid, formic acid, hydrobromic acid, sulfiuric acid, nitric acid, aceticacid, and the like.

[0103] Typically, the pH should be lowered to a value from about 5.5 toabout 6.5. If the pH is kept between 5.5 and 6.5, the9,10-epoxy-18-hydroxyoctadecanoic acid will not readily decompose to9,10,18-trihydroxyoctadecanoic acid. In addition, the yield of9,10-epoxy-18-hydroxyoctadecanoic acid decreases if the pH is notcarefully controlled. For example, if the pH falls below 4.0, the9,10-epoxy-18-hydroxyoctadecanoic acid is hydrolyzed to thecorresponding diol in less than two hours at room temperature.

[0104] As illustrated in FIG. 4, Step 7, the9,10-epoxy-18-hydroxyoctadecanoic acid precipitate (i.e., oxirane) isseparated from the fifth solution. The the oxirane can be separated fromthe fifth solution using any suitable technique. For example, theoxirane can be separated by filtering, hot filtering, or centrifuging.Specifically, the oxirane can be separated by filtering, as illustratedin the Examples herein below.

[0105] The oxirane can optionally be purified using any suitabletechnique known in the art. For example, the oxirane can be purified byrecrystallization, extraction, chromatography or sublimation.Specifically, the oxirane can be purified by recrystallization from analcohol (e.g., isopropanol), as illustrated in the Examples hereinbelow.

[0106] Isolation of 9,10,18-Trihydroxyoctadecanoic Acid from Outer BirchBark (FIG. 5)

[0107] As illustrated in FIG. 5, step 1, outer birch bark is subject toalkali hydrolysis as described herein above in FIG. 4, step 1.

[0108] Lupeol and betulin are optionally removed from the outer birchbark prior to the outer birch bark being subject to alkali hydrolysis tofacilitate the isolation of 9,10,18-trihydroxyoctadecanoic acid fromouter birch bark.

[0109] As illustrated in FIG. 5, step 2, the second solution isseparated from the second outer birch bark as described herein above inFIG. 4, step 2.

[0110] As illustrated in FIG. 5, step 3, the second solution iscondensed as described hereinabove in FIG. 4, step 3 to form a thirdsolution.

[0111] As illustrated in FIG. 5, steps 4 and 5, water is added to thethird solution to form a first precipitate and a fourth solution. Thefirst precipitate is then separated from the fourth solution asdescribed herein above in FIG. 4, step 5.

[0112] As illustrated in FIG. 5, step 6, the fourth solution isacidified to a pH of about 5.5 to about 6.5 as described herein above inFIG. 4, step 6 to provide a fifth solution and a second precipitate.

[0113] As illustrated in FIG. 5, step 7, the second precipitate isseparated from the fifth solution as disclosed herein above in FIG. 4,step 5.

[0114] The second precipitate can optionally be crystallized orprecipitated from a suitable solvent (e.g., an alcohol) to give a solidand a filtrate and the solid may be separated (e.g., filtered) from thefiltrate.

[0115] As illustrated in FIG. 5, step 8, the fifth solution is condensedas described herein above in FIG. 4, step 3 to provide a sixth solution.

[0116] As illustrated in FIG. 5, step 9, the sixth solution is subjectto epoxidizing conditions to provide an epoxide. The epoxide is thenhydrolyzed. Both reactions can conveniently be carried out in a singlereaction vessel. The sixth solution is subject to epoxidizing conditionsand the resulting epoxide is hydrolyzed under any suitable conditionsknown in the art. E. Seoane and M. Arno, Total Synthesis andStereochemistry of Phloionolic acids, Anales de Quimica, 73, N11,1336-1339 (1977). For example, the sixth solution may be epoxidized andhydrolyzed by the addition of hydrogen peroxide and an acid to the sixthsolution and subsequent heating of the resulting mixture.

[0117] As illustrated in FIG. 5, Step 10, 9,10,18-trihydroxyoctadecanoicacid is crystallized from the seventh solution. The crystalline productis then separated from solution. The crystalline product may beseparated from the seventh solution using any suitable technique knownin the art. For example, the crystalline product can be separated fromsolution by filtering, hot filtering or centrifuging. Specifically, the9,10,18-trihydroxyoctadecanoic acid is separated by filtering.

[0118] The 9,10,18-trihydroxyoctadecanoic acid can optionally bepurified using any suitable technique known in the art. For example,9,10,18-trihydroxyoctadecanoic acid can be purified byrecrystallization, extraction, chromatography or sublimation.Specifically, 9,10,18-trihydroxyoctadecanoic acid can be purified byrecrystallization from an aqueous alcohol solution (e.g., ethanol:water,90:10).

[0119] Isolation of Non-Soluble Polyphenolic Polymers and Fatty Acids(FIG. 6)

[0120] As used herein, “non-soluble polyphenolic polymers” are polymers,which are non-soluble in water at a pH below about 4.0, but aretypically soluble in water at a pH above about 6.0. In addition,non-soluble polyphenolic polymers are soluble in acetone, alcohols andother polar solvents.

[0121] As illustrated in FIG. 6, step 1, outer birch bark is subject toalkali hydrolysis as described herein above in FIG. 4, step 1 to providea second birch bark and a second solution.

[0122] Lupeol and betulin are optionally removed from the outer birchbark prior to the outer birch bark being subject to alkali hydrolysis tofacilitate the isolation of non-soluble polyphenolic polymers and fattyacids from outer birch bark.

[0123] As illustrated in FIG. 6, step 2, the second solution isseparated from the second outer birch bark as described herein above inFIG. 4, step 2.

[0124] As illustrated in FIG. 6, step 3, water is added to the secondouter birch bark to provide a third solution and a third outer birchbark.

[0125] As illustrated in FIG. 6, step 4, the third outer birch bark isseparated from the third solution as described herein above in FIG. 4,step 5.

[0126] As illustrated in FIG. 6, step 5, the third solution is acidifiedto a pH of about 3.0 to about 4.0 to give a fourth solution and amixture of non-soluble polyphenolic polymers and fatty acids(hereinafter “NPPP”). The pH of the solvent may be lowered by adding asuitable acid. Acceptable acids include, for example, hydrochloric acid,sulfuric acid, phosphoric acid, formic acid, hydrobromic acid, nitricacid, acetic acid and the like.

[0127] As illustrated in FIG. 6, step 6, the mixture of non-solublepolyphenolic polymers and fatty acids is separated from the fourthsolution as disclosed herein above in FIG. 5, step 10.

[0128] The mixture of fatty acids and non-soluble polyphenolic polymerscan be purified using any technique known in the art. For example, themixture of non-soluble polyphenolic polymers and fatty acids can bepurified by recrystallization, extraction, chromatography orsublimation.

[0129] Using the methods of the present invention, the yield of fattyacids and non-soluble polyphenolic polymer obtained is about 20 wt.% toabout 40 wt.% from the outer birch bark.

[0130] Isolation of Soluble Polyphenolic Polymers and Fatty Acids fromOuter Birch Bark (FIG. 7)

[0131] As used herein, “soluble polyphenolic polymer” are that portionof the polyphenolic polymer fraction dissolved in the media under aspecific set of conditions (e.g., solvent, temperature, pH, ionicstrength, etc.). Specifically, soluble polyphenolic polymers are solublein water in both acidic and basic conditions.

[0132] As illustrated in FIG. 7, step 1, outer birch bark is subject toalkali hydrolysis as decsribed herein above in FIG. 4, step 1 to providea second outer birch bark and a second solution.

[0133] Lupeol and betulin are optionally removed from the outer birchbark prior to the outer birch bark being subject to alkali hydrolysis tofacilitate the isolation of soluble polyphenolic polymers and fattyacids from outer birch bark.

[0134] As illustrated in FIG. 7, Step 2, the second outer birch bark isseparated from the second solution as decribed herein above in FIG. 4,step 2.

[0135] As illustrated in FIG. 7, step 3, water is added to the secondouter birch bark as described herein above for FIG. 6, step 3 to providea third outer birch bark and a third solution.

[0136] As illustrated in FIG. 7, step 4, the third solution is separatedfrom the third outer birch bark as described herein above in FIG. 7,step 4.

[0137] As illustrated in FIG. 7, step 5, the third solution is acidifiedto a pH of about 3.0 to about 4.0 as described herein above in FIG. 6,step 5 to give a fourth solution and a solid.

[0138] As illustrated in FIG. 7, step 6, the solid is separated from thefourth solution as disclosed herein above in FIG. 4, step 5.

[0139] As illustrated in FIG. 7, Step 7, an alcohol is added to thefourth solution to provide a fifth solution and a precipitate.

[0140] As illustrated in FIG. 7, Step 8, the fifth solution is separatedfrom the precipitate as disclosed herein above in FIG. 4, step 5.

[0141] As illustrated in FIG. 7, Step 9, the fifth solution is condensedas described herein above in FIG. 4, step 3 to provide a mixture ofsoluble polyphenolic polymers and fatty acids.

[0142] The mixture of soluble polyphenolic polymers and fatty acids canoptionally be purified using any suitable technique known in the art.For example, the mixture of soluble polyphenolic polymers and fattyacids can be purified by recrystallization, extraction, chromatographyor sublimation.

[0143] The yield of fatty acids and soluble polyphenolic polymersobtained from the processes of the invention is typically about 5 wt.%to about 25 wt.% from the outer birch bark. Ideally, the yield of fattyacids and soluble polyphenolic polymers obtained is about 12 wt.% toabout 18 wt.% from the outer birch bark.

[0144] The present invention will be described by the followingexamples. The examples are for illustration purposes and do nototherwise limit the invention.

EXAMPLES Example 1

[0145] Dry Method Of Outer Birch Bark Manufacturing

[0146] Birch bark (20 kg) from a drum debarker was air dried (24 hours,room temperature) such that the water content was less than 10% and wassubsequently fed into a YardMan Model 246-648D401 chipper/shredder withan 8 HP gas powered motor. The outer bark shreds and inner bark pieces(combined mass of 19.9 kg) were separated on a wire screen with openingsof {fraction (1/4)}-by-{fraction (1/4)}-inch. Outer bark shreds(approximately 5.0 to 6.9 kg) and inner bark chunks (approximately 13.0to 14.9 kg) were recovered from the screening process. The shreddedouter bark was reduced in size for extraction using a 15 HP 3B JuniorHarnmermill made by Jay Bee Manufacturing, Inc.

Example 2

[0147] Betulin, Betulinic Acid And Lupeol Manufacturng

[0148] Dried outer birch bark shreds (1000 g) were loaded in 5 literfractional supercritical fluid extraction vessel. The fractionalsupercritical fluid extraction was conducted by Phasex Corporation, 360Merrimack Street, Lawrence, Mass. 01843. The first supercritical fluidextraction was conducted at 45° C. and 4000 psi for two hours, employingcarbon dioxide as a solvent. After which time, the first fraction (60grams) was gathered in a separation vessel. The second supercriticalfluid extraction was conducted at 90° C. and 9000 psi for 4 hours,employing carbon dioxide as a solvent. After which time, the secondfraction (150 grams) was gathered in a separation vessel.

[0149] GC/MS analysis of the first fraction: 33% lupeol, 61% betulin, 2%betulinic acid, 4% other triterpenes and fatty acids.

[0150] GC/MS analysis of the second fraction: 2% lupeol, 80% betulin,13% betulinic acid, 5% other triterpenes and fatty acids.

[0151] The first fraction (60 g) was boiled with ethanol (1.2 liters)and aluminum isopropoxide (5 g) was added. The resulting mixture was hotfiltered and the filtrate was cooled at 0° C. for 3 hours. Crystalsformed from solution and were filtered to afford betulin (30 g, greaterthan 90% pure, yield 3% from dry bark).

[0152] The above filtrate was purified on silica [eluent: hexane-ether(4:1)] employing column chromatography at atmospheric pressure. Lupeol(17 g, 1.7 wt.% on dry bark, greater than 90% pure) and betulin (5 g,greater than 90% pure, 0.5 wt.% on dry bark) were obtained.

[0153] The second fraction (150 g) was boiled with ethanol (3 liters)and aluminum isopropoxide (20 g) was added. The resulting mixture washot filtered and the filtrate was evaporated to a volume 0.7 liters andcooled at 0° C. for 3 hours. Crystals fromed from solution and werefiltered to afford betulin (115 g, greater than 90% pure, yield 11.5%from dry bark).

[0154] Total yield: betulin: 3%+0.5%+11.5%=15% (from dry bark)

[0155] Total yield: lupeol: 1.7% (from dry bark)

[0156] Betulinic Acid Separation

[0157] a) The solids obtained from the above hot filtration of the firstfraction, ethanol, and aluminum isopropoxide were combined, washed withhot ethanol, acidified with 2% HC1, filtered, washed with hexane, washedwith hexane-ether (1:1), methylated with dimethylsulfide, purified onsilica [eluent:hexane-ether (4:1)] and hydrolyzed in 5% sodium hydroxidein ethanol to afford betulinic acid (16 g, greater than 95% purity,yield 1.6 wt.% on dry bark).

[0158] b) Alternatively, the solids obtained from the above hotfiltration of the first fraction, ethanol, and aluminum isopropoxidewere combined, washed with hot ethanol, acidified with 2% HC1, filtered,washed with hexane, washed with hexane-ether (1:1), washed withmethylene chloride, and recrystallized from methanol to providebetulinic acid (14 g, greater than 90% purity, yield 1.4 wt.% on drybark).

[0159] The betulin, betulinic acid and lupeol obtained from the methodsof the present invention were identical to commercial reagents and wereconfirmed by m.p, IR—, H¹—NMR and C¹³-NMR and GC/MS-spectra (fromAldrich Co., in Catalog 1997: Betulin-#12,376-5, p. 166; BetulinicAcid-#85,505-7; from Sigma Co., in Catalog 1999, Lupeol-L 5632, p. 655).

Example 2

[0160] Alkali Hydrolysis Of Birch Bark, Isolation Of22-Hydroxydocosanoic Fraction and 9,10-Epoxy-18-Hydroxyoctadecanoic AcidFraction

[0161] Outer birch bark (790 g) obtained after supercritical fluidextraction was added to a solution of NaOH (176 g, 4.4 mol) in 95%isopropanol (8 liters) and the mixture was refluxed (1 hour). After hotfiltration, isopropanol (3.8 liters) was added to the bark and themixture was refluxed (20 minutes). The reaction mixture was filtered andthe filtrate was evaporated in vacuo at 30° C. (GS/MC sample 1). H₂O (5liters) was added to the condensed residue and the mixture was stirredfor 2 hours at room temperature. The insoluble material was separated bycentrifugation and acidified with 6% HC1 to pH=4.7 (GS/MC sample 2) toafford 22-hydroxydocosanoic (90 g). The purity of the22-hydroxydocasanoic by GC/MS analysis is greater than 50%. The watersolution after centrifugation (i.e., supernatant) was acidified with 6%HC1 to pH=5.5-6.5 and the 9,10-epoxy-18-hydroxyoctadecanoic acidfraction (179 g) was obtained by filtration. The purity of9,10-epoxy-18-hydroxyoctadecanoic fraction by GCIMS analysis is greaterthan 70%.

Example 3

[0162] Recrystallization Of 9,10-Epoxy-18-Hydroxyoctadecanoic Acid

[0163]9,10-Epoxy-18-hydroxyoctadecanoic acid (179 g) was added toisopropanol (1.7 liters) and the mixture was allowed to reflux until allof the acid was dissolved. The temperature of the solution was decreasedto room temperature and precipitation occurred over a period ofapproximately 5 hours. After centrifuging, the solution was evaporatedand the resulting solid was crystallized in isopropanol (1 liter). Thecombined solids were dried in vacuo and9,10-epoxy-18-hydroxyoctadecanoic acid (81 g) was obtained. The purityis greater than 95% (GC/MS analysis).

Example 4

[0164] Isolation Of 9,10,18-Trihydroyoctadecanoic Acid (Pholiolic Acid)

[0165] The supernatant from Example 3 was evaporated and added to asolution of 30% H₂O₂ (164 ml, 1.4 mol H₂O₂) in 95% formic acid (1.3liters) and stirred for 3 hours at 40° C. The mixture was evaporated invacuo and a solution of 10% NaOH was added (to pH=9.0). The solution wasstirred for 1 hour at 60° C. and 6% aqueous HCI solution was addeddropwise to the stirring mixture (to pH=5.5). The resulting solids wereseparated by centrifugation, and dried in vacuo. The solids werecrystallized from ethanol-water (4/1) to afford9,10,18-trihydrohyoctadecanoic acid (96 g). The purity is greater than95% (GC/MS analysis).

Example 5

[0166] Polyphenolic Polymer And Fatty Acids Separation

[0167] The remaining bark (57 g) from alkaline hydrolysis in Example 2was added to water (7 liters) and stirred for 2 hours. The remainingsolids (112 g) were separated by centrifugation and the aqusous solutionwas acidified with 37% HC1 (to pH=3.0). The resulting mixture ofnon-soluble polyphenolic polymers (200 g) and fatty acids was isolatedvia centrifugation. The aqueous solution separated during centrifugationwas evaporated to a volume of 1 liter and ethanol (3 liters) was added.The solvent was evaporated and a mixture of fatty acids and solublepolyphenolic polymer (SPPP) was obtained. Yield 105 g (10 wt.%) on drybark.

[0168] All publications, patents, and patent documents are incorporatedby reference herein, as though individually incorporated by reference.The invention has been described with reference to various specific andpreferred embodiments and techniques. However, it should be understoodthat many variations and modifications may be made while remainingwithin the spirit and scope of the invention.

What is claimed is:
 1. A process for separating outer birch bark frominner birch bark comprising subjecting birch bark to fragmentation toprovide a combination of outer birch bark shreds and inner birch barkchunks and separating the outer birch bark shreds from the inner birchbark chunks.
 2. The process of claim 1 wherein the separating comprisespushing the outer birch bark shreds through a mesh effective to separatethe outer birch bark shreds from the inner birch bark chunks or theseparating is accomplished with the use of an air classifier.
 3. Theprocess of claim 1 or 2 wherein the fragmentation is accomplished with achipper or a shredder.
 4. The process of claim 1 or 2 further comprisingreducing the size of the outer birch bark shreds with the use of ahammermill.
 5. A process for obtaining a natural product from outerbirch bark comprising subjecting the outer birch bark to supercriticalfluid extraction to provide the natural product.
 6. The process of claim5 wherein the natural product is betulin, betulinic acid or lupeol. 7.The process of claim 5 wherein the supercritical fluid extractionutilizes carbon dioxide as a solvent.
 8. A process for obtaining lupeol,betulinic acid and betulin from outer birch bark comprising: extractingwith carbon dioxide at a pressure between about 3,000 psi and 10,000 psiand at a temperature between about 50° C. and 100° C. to provide lupeol,betulin and betulinic acid.
 9. A process for obtaining lupeol, betulinicacid and betulin from outer birch bark using fractional supercriticalfluid extraction comprising: extracting with carbon dioxide at apressure below about 5,000 psi and at a temperature below about 50° C.to provide a product comprising lupeol; and extracting with carbondioxide at a pressure of about 5,000 psi to about 10,000 psi and at atemperature of about 50° C. to about 120° C. to provide a productcomprising a mixture of betulin and betulinic acid.
 10. The process ofclaim 9 further comprising separating the betulin from the mixture ofbetulin and betulinic acid.
 11. A process for obtaining lupeol fromouter birch bark comprising: subjecting the outer birch bark tosupercritical fluid extraction with carbon dioxide at a temperature ofabout 40° C. to about 50° C. and a pressure of about 3,000 psi to about5,000 psi for a period of time of about 1 hour to about 3 hours toprovide the lupeol.
 12. A process for isolating9,10-epoxy-18-hydroxyoctadecanoic acid from outer birch bark comprising:(1) subjecting the outer birch bark to alkali hydrolysis in an aqueousalcohol solution to provide a second outer birch bark and a secondsolution; (2) separating the second solution from the second outer birchbark; (3) condensing the second solution at a temperature below about50° C. to form a third solution; (4) adding water to the third solutionto form a precipitate and a fourth solution; (5) separating theprecipitate from the fourth solution; (6) acidifying the fourth solutionto a pH of about 5.5 to about 6.5 to give a fifth solution and9,10-epoxy-18-hydroxydecanoic acid as a precipitate; and (7) separatingthe 9,10-epoxy-18-hydroxydecanoic acid precipitate from the fifthsolution to give 9,10-epoxy-18-hydroxydecanoic acid.
 13. The process ofclaim 12 wherein lupeol, betulin and betulinic acid are removed from theouter birch bark prior to the alkali hydrolysis.
 14. The process ofclaim 12 further comprising recrystallizing the9,10-epoxy-18-hydroxydecanoic acid from isopropanol, methanol orethanol.
 15. A process for isolating 9,10,18-trihydroxyoctadecanoic acidfrom outer birch bark comprising: (1) subjecting the outer birch bark toalkali hydrolysis in an aqueous alcohol solution to provide a secondouter birch bark and a second solution; (2) separating the secondsolution from the second outer birch bark; (3) condensing the secondsolution at a temperature below about 50° C. to form a third solution;(4) adding water to the third solution to form a first precipitate and afourth solution; (5) separating the first precipitate from the fourthsolution; (6) acidifying the fourth solution to a pH of about 5.5 toabout 6.5 to give a fifth solution and a second precipitate; (7)separating the second precipitate from the fifth solution; (8)condensing the fifth solution to provide a sixth solution; (9)subjecting the sixth solution to epoxidizing conditions to provide anepoxide and hydrolyzing the epoxide to provide a seventh solution; and(10) crystallizing the seventh solution to give9,10,18-trihydroxyoctadecanoic acid.
 16. The process of claim 15 whereinlupeol, betulinic acid and betulin are removed from the outer birch barkprior to the alkali hydrolysis.
 17. The process of claim 15 furthercomprising recrystallizing the 9,10,18-trihydroxyoctadecanoic acid froman alcohol or an aqueous alcohol solution.
 18. A process for isolatingnon-soluble polyphenolic polymers and fatty acids from outer birch barkcomprising: (1) subjecting the outer birch bark to alkali hydrolysis inan aqueous alcohol solution to provide a second birch bark and a secondsolution; (2) separating the second solution from the second outer birchbark; (3) adding water to the second outer birch bark to provide a thirdsolution and a third outer birch bark; (4) separating the third solutionfrom the third outer birch bark; (5) acidifying the third solution to apH of about 3.0 to about 4.0 to give a fourth solution and a mixture ofnon-soluble polyphenolic polymer and fatty acids; and (6) separating themixture of fatty acids and non-soluble polyphenolic polymers from thefourth solution.
 19. The process of claim 18 wherein lupeol, betulinicacid and betulin are removed from the outer birch bark prior to thealkali hydrolysis.
 20. A process for isolating fatty acids and solublepolyphenolic polymers from outer birch bark comprising: (1) subjectingthe outer birch bark to alkali hydrolysis in an aqueous alcohol solutionto provide a second outer birch bark and a second solution; (2)separating the second solution from the second outer birch bark; (3)adding water to the second outer birch bark to provide a third outerbirch bark and a third solution; (4) separating the third solution fromthe third outer birch bark; (5) acidifying the third solution to a pH ofabout 3.0-4.0 to give a fourth solution and a solid; (6) separating thesolid from the fourth solution; (7) adding an alcohol to the fourthsolution to provide a fifth solution and a precipitate; (8) separatingthe precipitate from the fifth solution; and (9) condensing the fifthsolution to provide a mixture of fatty acids and soluble polyphenolicpolymers.
 21. The process of claim 20 wherein lupeol, betulinic acid andbetulin are removed from the outer birch bark prior to the alkalihydrolysis.